Synthetic, plant and plasma antiproteinases inhibit the natural cytotoxicity (NK) of human peripheral blood lymphocytes to tumor cells. The role of proteinases in NK will be studied by in vitro substrate competition of NK and by identification of proteinases selectively active during NK. Class and substrate specificities of the proteinases will be determined by substrate competition of NK and by use of class specific inhibitors. Identification and characterization of NK-specific proteinases will be made by radiolabeling of proteinases, and examination to see if they are unique, or in elevated concentration, during NK. Proteinases will be labeled with 3H-DFA, or by 125I after isolation of plasma antiproteinase-proteinase complexes formed during NK. Characterization will include determination of m.wt. and isoelectric focusing point, localization in the NK incubation medium or in subcellular components, and approximate quantitation by specific radioactivity and microKjeldahl N analysis. The plasma alpha 1 antiproteinases that are most potent in inhibition of NK will be isolated and identified. These inhibitors will be used as probes to identify and isolate the proteinases involved in cytotoxicity. This study will contribute to understanding of the mechanism of cell-mediated cytotoxicity.